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rhPCR SNP genotyping method provides calls with >99.5% accuracy

Easy-to-use rhAmp™ SNP Genotyping System

The rhAmp™ SNP Genotyping System is a new genotyping platform developed with IDT proprietary RNase H2-dependent PCR technology. This technology uses a unique two-enzyme system coupled with RNA-DNA hybrid primers to precisely interrogate target SNPs. Combined with IDT universal reporter chemistry, the rhAmp SNP Genotyping System offers a simple, single-tube, high performance genotyping solution at an affordable price (Figure 1).

  • Generate the highest level of performance with greater than 99.5% call accuracy for over 90% of assays tested
  • Interrogate SNPs in difficult sequence regions with amplicons sizes as small as 40 bp
  • Validate markers affordably using the smallest pack size commercially available
  • Ensure confidence in your data with gBlocks® Gene Fragments as control templates
  • Get results quickly with assays shipped within 7 days from order

Figure 1. Simple, single-tube reaction chemistry supports streamlined lab processes. All reagents are combined in the initial reaction setup that is stable for up to 24 hr at room temperature before cycling. rhAmp™ SNP Genotyping is compatible with common qPCR platforms.

Blocked primers minimize non-specific amplification

With rhAmp™ technology, unique primer and enzyme features provide superior SNP discrimination over traditional SNP detection systems. One of the rhAmp primers contains an RNA base near its blocked 3′ end. The blocking group prevents primer-dimer artifacts and non-specific amplification that can cause unbalanced consumption of reaction components. During the annealing step, the RNase H2 enzyme recognizes the RNA base, when the RNA base is hybridized to its DNA complement, resulting in primer cleavage by the enzyme, and removal of the 3' blocking group. Only after this deblocking occurs is the primer available for extension by a novel Taq DNA Polymerase (IDT) with enhanced sensitivity for allelic mismatches. A tail sequence, included in the primers, is incorporated into the amplicon after the second cycle of amplification and is bound by the universal reporter probe. Signal is generated during extension and reporter probe degradation. Figure 2 summarizes these steps.

Figure 2. An rhAmp™ SNP Genotyping PCR cycle. All components needed to measure both alleles are combined in a single reaction before cycling. 1) Both allele-specific primers query the SNP locus. 2) RNase H2 enzyme cleaves the primers that are perfectly annealed to the target sequence, removing the RNA base and 3′ blocking modification, which allows extension by the novel, mismatch-sensitive Taq Polymerase (IDT). 3) During the first 2 amplification cycles, a tail sequence is incorporated into the amplicon that is subsequently recognized by a probe-based reporter system. 4) Polymerase extension leads to degradation of the probe and signal generation.

SNP discrimination—rhAmp™ vs 5′ nuclease assay

rhAmp SNP Assays deliver increased signal-to-noise ratios over traditional 5′-nuclease chemistry. The assay format uses allele-specific primers with a universal reporter system for signal generation. The assay design does not need to accommodate the use of a probe overlaying the SNP, allowing for smaller amplicon sizes, thus, facilitating assay designs in challenging genomic locations. The rhAmp universal reporter sequences are optimized to minimize interaction with the human genome, and provide robust and consistent signal. These features lead to improved cluster separation for higher confidence genotyping calls. Figure 3 compares data from rhAmp and 5′-nuclease assays for the detection of a SNP located in the ADME (absorption, distribution, metabolism, and excretion) gene, CYP1A2, in 91 human genomic DNA samples. Results, displayed in the allelic discrimination plot demonstrate the higher fluorescent signal and improved cluster separation provided by rhAmp genotyping.

Figure 3. The rhAmp™ SNP Genotyping System achieves higher fluorescent signal and improved cluster separation compared to assays from other manufacturers. In rhAmp technology, primer-dimer formation and non-specific amplification are prevented, thus eliminating unbalanced consumption of reaction components. Moreover, rhAmp universal reporter sequences are optimized to provide robust and consistent signal. Here, an allelic discrimination plot for a SNP located in the ADME (absorption, distribution, metabolism, and excretion) gene, CYP1A2 (rs762551), shows higher fluorescent signal for rhAmp ADME Assays (blue circles) than 5′ nuclease assays from Supplier T (green circles). Human genomic DNA (gDNA) from 91 individuals (Coriell Institute) was analyzed using 3 ng of gDNA per 5 µL reaction. Analysis was performed using QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher).

Complete genotyping solution

The rhAmp SNP Genotyping System portfolio includes all the components needed to successfully generate high quality genotyping data, including the largest collection of human predesigned genotyping assays, covering more than 10 million validated SNPs.

  • Assays
    • rhAmp SNP Assays are universal reporter–based SNP genotyping assays with RNase H2-cleavable primers for improved accuracy and specificity. Predesigned assays are available for human SNPs, or may be custom designed using the rhAmp™ Genotyping Design Tool.
    • rhAmp ADME SNP Assays are predesigned and experimentally validated assays targeting genes responsible for the absorption, distribution, metabolism, and excretion (ADME) of pharmaceutical compounds.
  • Ready-to-use mixes
    • rhAmp Genotyping Master Mix contains the required enzymes and components necessary for activation and amplification of rhAmp SNP assays.
    • rhAmp Reporter Mixes are universal reporter probes mixes, available with or without the reference dye required by certain instruments.
  • Mismatch-sensitive Taq Polymerase
    • A uniquely modified enzyme with increased sensitivity toward allelic mismatches.
  • Controls
    • gBlocks Gene Fragments are double-stranded, sequence-verified DNA blocks that can be used as synthetic controls and are easily available through an integrated ordering process.
  • Design tool
    • The rhAmp Genotyping Design Tool facilitates assay designs for SNPs not currently included in our human predesigned assay library or for SNPs in any other species.

Learn more about the rhAmp SNP Genotyping System at www.idtdna.com/rhAmp-genotyping.

Related reading

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Author: Sharon Rouw is a marketing manager at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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